ABSTRACT - PROJECT 3 Metalloproteins that contain a heme prosthetic group (hemeproteins) are essential to lung functions, including airway host defense and inflammation [inducible nitric oxide (NO) synthase (iNOS)], and bronchomotor tone [soluble guanylate cyclase (sGC)]. We recently discovered that NO inhibits heme insertion into apo-proteins by the S-nitrosyation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which functions in heme transport and insertion. Recycling GAPDH is dependent on cellular redox and S-denitrosylase enzymes, i.e. GSNO reductase (GSNOR) or thioredoxin 1 (Trx1). These exciting findings and longstanding interactions with Program members in the study of asthma over the years made it a natural step to investigate whether the high- levels of NO in the asthmatic airway blocks hemeprotein maturation. Our preliminary results show that asthmatic airway epithelium escapes NO-mediated inhibition of heme insertion by upregulation of denitrosylase pathways, enabling the expression of active iNOS, but that airway smooth muscle cells are overwhelmed by NO at the levels produced in the asthmatic airway, with consequent loss of heme-replete sGC. New preliminary data generated in murine models of asthma and in the human precision cut lung slice model suggest that heme-independent sGC-activators are strikingly effective bronchodilators. Thus, we will test the hypothesis that thiol denitrosylase systems enable continuous airway epithelial NO production by iNOS in asthma by restoration of hemeprotein maturation, but that the NO overwhelms smooth muscle denitrosylase systems, compromising maturation & function of the sGC in smooth muscle, which results in bronchoconstriction and airway hyper-reactivity. We plan concurrent mechanistic studies and studies of the bronchodilator response to novel pharmacologic sGC agonists in (i) primary airway smooth muscle cells from healthy and asthmatic airways, (ii) precision cut human lung slice model and (iii) mouse models of asthma using genetically modified mice (including NOS1,2,&3 -/-, GSNOR-/- and smooth muscle specific sGC-/-). Also, we will study hemeprotein maturation in airway epithelial cells freshly obtained from healthy individuals and asthmatics with low or high FENO to help verify clinical relevance and potentially endotype sGC-agonist responders. sGC agonists will be tested in order to translate the discoveries to patient care soon. This research will provide the first information on hemeprotein maturation in healthy and asthmatic airway, and test connections between dysregulation of hemeprotein maturation and the bronchoconstriction and inflammation characteristic of asthma.